Jurkat rejection of R5-HIV infection. - (Nov/11/2006 )
Greetings all ^_^,
I've been performing in vitro infection of T-cells for a while now in a particular project. Primary cells are induced to express CCR5 at high levels. FACS is used to confirm this high expression. CCR5 cells are then infected with a GFP labeled R5-HIV at MOI's between 1 and 3. Most of these primary cells are sucessfully infected and express GFP at high levels when infected within 2-3 days. All cells are cultured in 10% serum RPMI with MEM Viatims, MEM ammino acids, Sodium pyruvate, Pen/Strep antibacterial, and L-glutamine.
Recently, due to difficulties with cell death, primary T-cells were replaced with the Jurkat transformed cell line. The same proceedures are followed. In every experiment performed with Jurkats, GFP expression by as late as day 6 has not risen, leading me to believe that the Jurkats are not being infected. The virus titer is around 8MIU/ml. Cells are cultured at 0.7M per well. MOI's are again between 1 and 3. Positive controls are also not taking up the virus. Experiments performed under the same conditions with the viral envelope (HIV viral packaging with GFP, no viral genome) induce high GFP expression within 3 days.
Thus the question, why won't my jurkats infect? I've used three different lines of Jurkats ranging from 5 days to 35 days old. Every line shows practically the same response. The only consistency is the CCR5 virus used to induce CCR5 expression. Could it be that? What am I doing wrong o_o?
working with CCR5 HIV as well, I've also tested a Jurkat CCR5+ cell line with results that weren't too good. I've subsequently done experiments with other cells and mostly (even though CCR5 expression can be quite high in certain transfected cells) infection is rather low, even though p24 values of the viral stocks are quite high. Reasons might be that CCR5 isn't located too close to CD4 or that the conformation of CCR5 isn't favorable for HIV-infection.
Can't explain your positive control though. Is the envelope you used for this control the same as the one used for "real" infection? There tends to be a big difference between infectivity for different HIV's, mostly env contributes to this...
Have you performed a control with a X4 virus? Might be some sort of problem inside your cell, not related to CCR5?